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Characterization studies of the wistar rat hepatic cytosolic methlytrienolone (R1881) binding protein

University of British Columbia

Age and sex differences in adult rat hepatic microsomal drug metabolism are well-known. Although the exact mechanism is unclear, the role of testosterone has been clearly implicated. Since androgen sensitive sex end-organ tissues are known to contain specific androgen receptors which mediate the action of testosterone in these tissues, a similar mechanism may be involved in testosterone sensitive hepatic microsomal drug metabolism. To test this hypothesis, testosterone sensitive liver microsomal benzo(a)pyrene hydroxylase activities and cytosolic androgen binding protein levels were measured in vitro, using different animal models. Methyltrienolone (R1881) was used as the ligand for the cytosolic androgen binding assay, since testosterone was significantly metabolized in vitro. Scatchard analysis of adult male rat liver whole cytosol indicated two R1881 binding isotherms: a high affinity-low capacity binding site, and a lower affinity-higher capacity component. Addition of excess triamcinolone or Cortisol to the assay mixture eliminated the lower affinity binding component. The high affinity component was not present in the immature female or the intact or the 10-day ovariectomized adult female rat; it was present in low quantities in the immature male. Inhibition studies of the high affinity component using different competitors indicated a high specificity to androgens, including testosterone, dihydrotestosterone, androstenedione, R1881 and mibolerone, a moderate specificity to cyproterone acetate, estradiol and flutamide hydroxide, and no specificity to progesterone, triamcinolone, diethylstilbestrol and Cortisol. Saturation analysis of the high affinity component indicated that testosterone, dihydrotestosterone, androstenedione, mibolerone, cyproterone acetate and estradiol produced similar binding kinetics, indicating that these steroids were binding to the same or a similar high affinity site. Flutamide hydroxide yielded displaceable but non-saturable binding. Castration of adult male rats for 18 hrs, 4 or 10 days, or hypophysectomy for 10-17 days did not have a significant effect on the high affinity binding protein kinetics compared to controls. Nor did testosterone administration to these animals alter this protein's binding kinetics. Flutamide or flutamide hydroxide treatment to testosterone-treated adult male rats significantly reduced both liver microsomal benzo(a)pyrene hydroxylase activities and high affinity binding protein levels compared to the flutamide untreated controls. Streptozotocin treatment to adult males caused a marked decrease in the high affinity binding protein levels. These results indicate that the presence of the high affinity component in the adult male rat is not dependent upon gonadal or pituitary hormones in vivo, but it is altered following flutamide, flutamide hydroxide or streptozotocin administration. These studies do not negate the hypothesis that the presence as well asf the agonist occupancy of the high affinity-low capacity androgen binding protein are prerequisites for testosterone-sensitive effects in the liver.

Text

2010-06-13

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http://hdl.handle.net/2429/25638

Pharmaceutical Sciences

University of British Columbia

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